Most current immunoassays for high concentration, high molecular weight analytes are difficult to run on widely used clinical chemistry analyzers that use colorimetric or chemiluminescent detection systems.
Typically, immunoassays for high concentration, high molecular weight analytes in the marketplace are predicated on the multivalence of the analyte. Ultimately, the analyte is detected by some sort of cross-linking, either by agglutination (in turbidimetric or nephelometric assays), precipitation (radial immunodiffusion), or sandwich immunoassays. These types of immunoassays each have significant handicaps in translation into automated systems. Radial immunodiffusion assays are extremely slow (hours to days) and require substantial quantities of carefully selected antisera. Agglutination-based assays require both initial dilution and substantial quantities of immunomaterials. In addition, each of these methods requires specialized optical systems not ordinarily present on contemporary clinical analyzers. Sandwich or two-site immunoassays require either large initial dilutions or undesirably large concentrations of expensive immunomaterials.
The current capability of the formats directed to competitive immunoassays is best applied to high concentration, low molecular weight analytes like many therapeutic drugs or drugs of abuse. Traditionally, these competitive immunoassays are predicated on competition for a limited number of drug specific binding sites (immobilized antibody molecules) between free drug and an enzyme label prepared by chemical conjugation of a drug-derived hapten and horseradish peroxidase (HRP). Typically, selection criteria for the reagents for these diagnostic tests might include: first, the Kd (dissociation constant) of the drug:antibody complex must be similar to (within a factor of 10) the concentration of drug in the serum sample; and second, the Kd of the label:antibody complex must be substantially lower (one to several orders of magnitude depending on the absolute concentration of the analyte) than that of the drug:antibody complex under the same conditions. A problem encountered lies in the difficulty in making antibodies and labels with the necessary affinity requirements.
Satisfying the above conditions for high concentration, high molecular weight analytes is difficult. In particular the second condition (higher affinity for the labeled analyte), listed above is difficult to achieve. For small molecules, like drugs, the affinity of the antibody for the label can be enhanced by several effects including the “linker effect” (additional bonding energy due to interaction of the antibody with the linker), multisubstitution of the label with the hapten, and, possibly, favorable orientation of the drug on the label surface. Equivalent effects for macromolecular analytes are unlikely because the epitope for interaction with the analyte and the analyte:enzyme conjugate are identical and reside entirely on the analyte. Put another way, the analyte looks the same to the antibody whether it is free in solution or conjugated to an HRP molecule.